A Review of Brucellosis in Iran: Epidemiology, Risk Factors, Diagnosis, Control, and Prevention

Brucellosis caused by species of Brucella is among the most prevalent zoonoses with the annual incidence of half a million cases globally. Most parts of Iran are endemic for brucellosis, and the annual incidence of the human and animal brucellosis is still high. At present, there is no safe and protective human vaccine against brucellosis, and the only preventive strategy is animal vaccination, which harbors significant disadvantages. Considering the identification of many immunogenic proteins in Brucella, several studies have recently been performed to evaluate the vaccine potency of such antigens as a new subunit vaccine candidate. This review represents an overview of brucellosis in Iran, including epidemiology, transmission routs, diagnosis, and treatment. Moreover, it mainly highlights the history of brucellosis control and prevention in Iran, including eradication programs, vast livestock vaccination programs, and subunit vaccine studies. It also discusses major problems that the country encounters with disease control. In recent years, Persian scientists have focused on evaluating the efficacy of best Brucella immunogens in vivo to introduce a new subunit vaccine. The results of some studies could demonstrate the vaccine potential of some immunogens.


INTRODUCTION
rucellosis is one of the most prevalent bacterial zoonotic diseases causing significant economic losses due to the livestock abortion, and also it is possibly a life-threatening multi-system disease in human [1,2] . In 1886, Brucella melitensis was first isolated by David Bruce from the spleen of a British soldier who had died of a febrile illness, which was known as Malta fever and it was common among military personnel stationed on the island of Malta. The bacterium was named Micrococcus melitensis, with 'melitensis' derived from the Roman name for Malta, 'Melita'. In 1897, Bacillus abortus was identified as the cause of contagious abortion in cattle by Bernhard Bang. Later, in 1917, it was found that the causes of the two diseases were identical, and renamed Brucella in honor of Bruce [3,4] . In Iran, B. melitensis was primarily isolated from the human blood culture in 1932, and in the cattle population, B. abortus was isolated from an aborted fetus in 1944 [5] .

Epidemiology
Annually, more than 500,000 new human cases of brucellosis are reported worldwide [1,19] . The prevalence rates of brucellosis are more than 10 cases per 100,000 population in some countries. However, it is believed that the incidence of disease is underestimated since for each reported case, 26 cases are not detected The incidence rate in endemic areas is under 0.03 to more than 200 in 100,000 persons [20,21] . Many parts of the world are still endemic for brucellosis, including the Middle East (Iran), Africa, Latin America, Central Asia, and the Mediterranean Basin. Most parts of Iran are endemic for the disease, especially the areas where human lives in a close contact with livestock [1,22,23] . According to the report of the Ministry of Health and Medical Education (as cited in Zeinali et al.'s [24] and Esmaeili's [25] works), based on the incidence of human brucellosis in Iran, provinces are categorized into four types (Table 2). Moreover, in a study on trends of human brucellosis between 1991 and 2008, the mean annual incidence of human brucellosis was reported as 43.24 per 100,000 population [26] . In a recent study by Rostami et al. [27] on 1698 patients from 30 provinces of Iran, the mean of brucellosis incidence was reported to be 29.83 in 100,000 population (55% males and 45% females). In another study, Kassiri et al. [28] indicated that the incidence of Brucellosis in West of Iran was 59.31 per 100,000 population (34.9% females and 65.1% males), and nearly 95.2% of human cases were living in rural and 4.8% in urban areas. Moreover, the incidence rate of brucellosis in children in this region of Iran was 41.4 per 100,000 population (68.9% boys and 31.1% girls), among which 87.8% resided in rural areas [29] . On the other hand, Bokaie et al.'s [30] report showed the incidence of brucellosis as 37 per 100,000 population in East of Iran. Regarding the prevalence of brucellosis by age, the median age of 31.3 years has been reported by Zeinalian Dastjerdi et al. [31] . The prevalence of brucellosis in men is higher than in women in industrialized countries; however, in Iran, due to the close cooperation of women with men in ranching and farming occupations, the disease is also highly frequent in women [32] . In Sofian's work [33] , 55.3% of the affected pateints were male and 44.7% were female (with the mean age of 33.37±21.3). Moreover, the global investigation of the seasonal pattern of brucellosis shows that the disease is more prevalent in the first half of the year, which is the livestock's offspring season [34] . The rate of brucellosis enhances during spring and summer due to factors such as direct contacts between ranchers and aborted fetuses as well as consumption of contaminated dairy products. In contrast, the rate of the disease decreases in the second half of the year. Determining seasonal pattern of Brucellosis in Iran using meta-analysis showed that the highest incidence of brucellosis occurred during spring and summer, while the lowest incidence occurred during winter and autumn [34] . Rostami et al. [27] also indicated that the highest and lowest cases of brucellosis are observed in spring and autumn, respectively. Table 2. Classification of Iran's provinces based on human brucellosis incidence [24,25]

Transmission and risk factors
The prevalence of human brucellosis is dependent upon factors such as husbandry practices, dietary habits, methods of processing milk, and dairy products, as well as environmental sanitation [10] . Brucellosis is transmitted from infected livestock to human via ingestion (unpasteurized milk or dairy products), inhalation, conjunctiva, or skin abrasions [22,35] . Brucellosis is usually considered as an occupational disease because it occurs mostly in abattoir workers, veterinarians, lab technicians, hunters, farmers, and livestock producers. The transmission of brucellosis is not usually from person to person; nevertheless, it may be transmitted via blood transfusion, bone marrow transplantation, sexual contact, or congenital [10,19,22] . Identifying the major risk factors for brucellosis is very important to reach a comprehensive understanding of the nature of the disease and its transmission routes for eradication of human brucellosis [33] . In Iran, the main risk factors for brucellosis are consumption of unpasteurized dairy products (especially raw milk and fresh cheese), direct contact with animals and animal husbandry, laboratory and veterinary professions, and the presence of another case of brucellosis at home [33,[36][37][38][39] . Furthermore, the geographic situation of Iran is considered as an important risk factor for the extension of contagious diseases, particularly from the Eastern and Western neighbor countries such as Iraq, Pakistan, and Afghanistan. Due to the lack of accurate control programs for animal diseases in these countries, brucellosis is endemic in these areas and therefore, there is a risk of brucellosis transmission from these countries to Iran [25] .

Clinical manifestations
Based on the course of the disease, human brucellosis is classified into three forms: (1) acute brucellosis characterized by weakness, undulant fever, headaches, myalgia, fine red rash, splenomegaly, hepatomegaly, and gastrointestinal symptoms. The acute phase may end in death, curing, transition into a sub-acute or chronic form; (2) sub-acute brucellosis characterized by almost all symptoms typical of the acute course but milder; (3) chronic brucellosis in which long-term signs and symptoms may include fatigue, recurrent fevers, arthritis, endocarditis, and spondylitis [7,22,41,42] . In a study performed by Beheshti et al. [37] , sweating, myalgia, and weakness were symptoms that were highly predictive for having a positive serological test result. Moreover, the prevalence of arthritis in children has been reported to be 24 out of 96 cases (25%) diagnosed with brucellosis [43] . The most common manifestations were fever (87.5%) and fatigue (75%). Three cases of Brucella infection of the thyroid gland were reported by Azizi and Katchoui [44] . All three cases were female, and two were from rural areas in Tehran Province.

Diagnosis
Microscopic examination of stained smears is a useful tool for probable diagnosis, especially if it is confirmed by other tests. Brucella are arranged as a single coccobacilli or short rods, though they are sometimes in pairs or small groups [23] . Although blood culture is known as the gold standard in the diagnosis of brucellosis, it has major limitations such as being time-consuming and needing biosafety level 3 and expert personnels [45] . Culture or serology is the definitive diagnostic methods for human brucellosis. Blood, tissue samples, pus and cerebrospinal, joint, ascitic, or pleural fluid can be used for the isolation of Brucella [10,46] . Automated culture systems, replacing the traditional biphasic Ruiz-Castaneda system, are now a safer and faster method of diagnosis [47,48] . According to the study performed by Purcell et al. [7] the detection rate of BACTEC (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD, USA), Myco/F Lytic medium in conjunction with BACTEC 9240 blood culture system was 80%. However, the detection rate of the pediatric Peds Plus/F or adult Plus Aerobic/F medium in conjunction with BACTEC 9240 blood culture system was 100% [7] . The sensitivity of both traditional and automated methods for the acute form had been reported to be about 90%. However, the sensitivity of the Biphasic Ruiz-Castaneda system in the chronic form is less than 20% in comparison to the sensitivity of 70% for the automated systems [49,50] . Traditionally, alternative brucellosis diagnostic methods in the absence of culture facilities are serological tests such as the Rose Bengal test, the serum agglutination test (SAT), and the antiglobulin or Coombs' test, which are based on the reactivity of antibodies against smooth lipopolysaccharide (LPS). Usually, the Rose Bengal test is used as a screening test, and positive samples are confirmed by the SAT. The sensitivity of the Rose Bengal plate test is more than 99%, and the rate of false-negative results is infrequent [7,10,19,48] . Enzyme-linked immune-sorbent assay (ELISA) that measures IgG, IgM, and IgA antibodies has advantages of having high sensitivity and possibility of better interpretation of the clinical situation. However, the specificity of ELISA is less in comparison to the agglutination tests [19,48] . Finally, molecular detection methods are rapid and convenient for the diagnosis of human brucellosis and may improve sensitivity relative to the culture. Brucella is detectable from serum, blood, pus, and tissue, but the usage of blood by PCR test is more usual. Although blood sample is more common for the molecular detection of human brucellosis, serum specimen is more popular and has priority over blood [51,52] . Several genus-specific multiplex PCR systems are developed based on primer pairs that target IS711, IS650, 16SRNA, BCPS31, and omp2a sequences. PCR can also be used for assessing the treatment efficacy, species differentiation, and biotyping of isolates [22,45,46] . The results of Alikhani et al.'s [53] study on the comparison of blood culture BACTECH system and whole blood and serum PCR method indicated that PCR can be considered as a sensitive and specific method for the diagnosis of human brucellosis. Moreover, in a study performed by Hajia et al. [54] , SAT, Coombs Wright test, 2-mercaptoethanol test (2ME), ELISA (IgG and IgM), and PCR method have been compared using serum samples. Among the applied methods of diagnosis, the SAT displayed the lowest positivity rate and ELISA test had the highest efficiency. Also, the sensitivity of the PCR method was lower in comparison to ELISA.

Treatment
Despite the application of WHO's antibiotic regimen recommendation (1986), which consists of doxycycline 100 mg orally twice a day for 6 weeks plus oral rifampicin 600 to 900 mg daily for 6 weeks or streptomycin 1 g intramuscularly daily for 2-3 weeks, the rate of brucellosis treatment failure and relapse has been increased between 5-15% cases. The choice therapeutic regimen for the uncomplicated brucellosis consists of streptomycin for 2 to 3 weeks plus doxycycline for 8 weeks or gentamicin for 5-7 days plus doxycycline for 8 weeks [55] . The second-line agents such as quinolones or trimethoprimsulfamethoxazole can be administered for patients with treatment failure or repeated relapses. For patients with a complicated disease, treatment intervention requires a careful evaluation of the patient and a thorough therapeutic plan. Patients with spondylitis should possibly receive a quinolone in the initial regimen, for a protracted period [56] . According to Kassiri et al. [28] , the treatment regimen of doxycycline plus rifampin was used in 60.4% of brucellosis cases. However, Haddadi et al.'s [57] study in Tehran showed that the combination of cotrimoxazole and doxycycline was more effective in disease control.

Control and prevention Control and eradication
In 1998, WHO suggested general strategies as well as the Mediterranean Zoonoses Control Program for the eradication of animal brucellosis. The strategies and program included (1) prevention of disease extension among animals and monitoring brucellosis-free herds and regions, (2) identification of infected animals using diagnostic tests and their elimination by slaughter programs to generate brucellosis-free herds and zones, and (3) applying vast vaccination programs to decrease the disease prevalence. However, pasteurization of dairy products is considered as a significant safety method in endemic areas. Consumption of unpasteurized milk and dairy products and also raw or undercooked animal products (including bone marrow) should be avoided. Occupational exposure to Brucella can be prevented by good hygiene and using protective clothing/equipment. The use of safety measures are essential to prevent skin contamination, inhalation, or accidental ingestion of organisms while assisting at the birth, carrying out a necropsy, or butchering an animal. Moreover, handling an aborted fetus or its membranes and fluids requires especial precaution [4,19,35,39] . As described previously, consumption of unpasteurized milk and dairy products, slaughtered meat, and direct contact with animals are the main risk factors of brucellosis in Iran. Thus, improved veterinary services and public health education may play an important role in the disease control [36] . According to Esmaeili [25] , there are numbers of major problems for brucellosis control in Iran that include: (1) lack of a proper law for the punishment of violators in animal health field, (2) weaknesses in border quarantine system and animal trafficking from neighboring countries, (3) lack of rural and nomadic livestock identification system, (4) nomadic and semi-nomadic conditions of small ruminant husbandry that make the control of animal movement very difficult, (5) keeping sheep more than the immunity period of Rev.1 vaccine in some areas, and (6) making low payment to veterinarians who fight against the disease in operation teams.

Live, attenuated vaccines
Since presently there is no safe and protective human vaccine against brucellosis, animal vaccination is a critical factor for the control and eradication of animal and human brucellosis. An ideal vaccine against Brucella should: (1) prevent Brucella infection in both genders, (2) not provoke disease in immunized animals, (3) prevent abortion, (4) confer long-term protection with only one dose, (5) not interfere with LPS-based serological tests, (6) be biologically stable and not present the risk of virulence reversion, (7) not be pathogenic to humans, and (8) not contaminate the derivatives of the vaccinated animals [58] . At present, animal vaccination against Brucella infection is usually performed by the administration of the live attenuated smooth Brucella strains, including B. abortus strain S19, B. abortus strain RB51, and B. melitensis strain Rev.1 [22] . Although live, attenuated vaccines promote long-term protection, they have major disadvantages: (1) causing abortion in pregnant animals, (2) secreting in milk of vaccinated animals, (3) being pathogenic to humans, (4) interfering with the LPS-based diagnostic tests, and (5) being resistant to rimfampicin, the first antibiotic of choice to human brucellosis treatment [22,59] .
Since 1960s, vaccination with B. melitensis strain Rev. 1 has been considered as the main strategy for the control of brucellosis in small ruminant in Iran. In 1963, early studies in the production of Rev.1 vaccine, as a domesticated biological vaccine, were started in Razi Vaccine and Serum Research Institute of Iran in cooperation with WHO. Since 1963, testing the vaccine efficacy in goats and sheep has proven that REV.1 vaccine is able to decrease the epidemic rate of the disease from 45% to 1.8% [25] . During 1983-2003, vaccination program was limited to young animals, and a test-and-slaughter campaign was conducted in adult sheep and goats using Rose Bengal, SAT, and 2ME tests. From 2003, control program was based on the mass vaccination of lambs and kids at the age of 4-7 months, using full doses (1-3×10 9 colony-forming units [CFU]) of Rev.1 vaccine, and also based on the immunization of the adult female animals with the reduced doses of the vaccine (0.5-2×10 6 CFU). Additionally, upon performing other control programs, including public education, promotion of sanitary husbandry practices, and microbiological assessment of herds with abortion outbreaks, the number of annual new human cases was reduced from 39 in 2005-2006 to 15.9 in 2010-2011 per 100,000 population [17] . The first vaccination program was performed for cattle in 1949. Because of the high prevalence of bovine abortion due to B. abortus, vaccination of adult cows and 3-8-month-old calves has been started using S19 vaccine since 1967 [60,61] . Adult vaccination using S19 vaccine was replaced by K45/20A in 1972, whereas vaccination with K45/20A was discontinued in 1980. Since 1988, all female cattle between 3-6 months of age have been vaccinated with S19, and hygiene education programs have been implemented for farmers. In 2007, S19 vaccine was removed from the brucellosis control program, and since then all cattle were immunized with RB51 [60,61] . In order to control the bovine brucellosis in dairy cow farms, the Iranian Veterinary Organization has recently set up a control program using a mass vaccination with RB51 and also test-and-slaughter and quarantine, as eradication measures. Subcutaneous vaccination of calves with 1-3.410 10 CFU of RB51 and the reduced dose of 1-3.410 9 CFU for adult animals are officially recommended [62] . However, in 2007 in Iran, cases of abortion in dairy cows were reported by Sharifi et al. [62] following vaccination with strain RB51 [62] . Pishva and Salehi [63] also reported the first isolation of B. melitensis vaccine strain REV.1 in cattle in Iran. The authors hypothesized that in traditional farms where cattle and sheep are kept in the same place, ewe vaccination (Rev.1) can be a source of Brucella infection and abortion in cattle.

Subunit vaccines
In the recent decades, subunit vaccines are becoming promising vaccine candidates against Brucella infection due to their safety profile. The major advantages of subunit vaccines over live vaccines is that subunit vaccines eliminate safety concerns associated with, they are less biohazardous, well defined, avirulent, noninfectious, and nonviable. However, subunit vaccines cannot replicate the immunogenicity of live vaccines and thus, they are not as effective as live attenuated vaccines. In order to develop a new effective Brucella vaccine, selection of an immunogen with potential to induce an adequate immune responses (biased towards a Th1) and confer the high level of protection is essential [4,58] . Recently, immunoproteomics approaches considerably facilitated 354 Iran. Biomed. J. 21 (6): 349-359  Table 3). The first world report on the evaluation of the protective efficacy of the 36 kDa outer membrane protein 2b (Omp2b) antigen and its truncated form has been performed by Golshani et al. [64,65] . Using immunoinformatics algorithms, mapping potential Tand B-cell epitopes are promising approaches to design new vaccine candidates. Furthermore, the numbers of immunogens have been analyzed using bioinformatics tools to design new vaccine targets based on epitope mapping [66][67][68][69][70][71][72] .
All parts of Iran are endemic for brucellosis, and it causes high economic loss due to livestock abortion and has serious public health consequences. Brucellosis has been an occupational risk for people having contact with infected animals, and nonoccupational source of the disease includes consumption of fresh and unpasteurized dairy products. The major problems for the control of brucellosis in Iran can be listed as following: -Lack of public knowledge about brucellosis, especially in rural areas -Public habit for consumption of raw milk and unpasteurized dairy products -Lack of proper eradication program for infected animals -Limitations of the commercially available animal vaccines -Lack of the protective and safe human vaccine -Keeping vaccinated livestock more than the protection period of the REV.1 vaccine -Lack of proper border quarantine system and infected animal trafficking from neighboring countries -Use of traditional livestock husbandry system in many rural areas -Lack of livestock identification system in rural and nomadic areas causing the lack of information about animals' immunization history -Construction of animal barn near human house -Lack of proper cooperation between farmers/livestock producers with veterinarians and Iran veterinary organization With regard to these facts, educating farmers and people living in the endemic areas, routine screening of domestic livestock, inhibiting animal trafficking from infected neighboring countries, eliminating infected animals, and setting up vast vaccination programs can decrease the risk of both animal and human infection. Vast immunization of livestock is the most preventive program in the endemic countries like Iran; however, due to the major limitations of the current commercial available vaccines and the lack of the human vaccine, finding a new protective and safe vaccine target seems to be essential.

CONFLICT OF INTEREST.
None declared.